This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Type III protein secretion systems (TTSSs) are essential virulence factors of many human and plant bacterial pathogens. These bacteria include some of the most important and devastating human and plant pathogens in the genera Yersinia, Salmonella, Escherichia, Shigella, Pseudomonas, and Xanthomonas. TTSSs are elaborate protein export devices that enable bacteria to inject virulence proteins, called effectors, directly into their eukaryotic host cells. Effector proteins are thought to travel this pathway in a largely unfolded manner and a set of customized cytosolic chaperones (TCC for Type III chaperones) are essential for secretion of a subset of the effectors. Although some chaperones of TTSS effectors have been intensively studied and characterized, their essential function remains a matter of controversy and studying this topic will promote our understanding of TTSSs and may allow for novel strategies to control plant and animal diseases caused by TTSS-containing pathogens. Type III effectors are multidomain proteins that contain N-terminal secretion and translocation signals and one or more C-terminal domains with host cell effector activities. The secretion signals are contained within the first 15-20 amino acids or codons. TTCs generally bind to a 50 -100 amino acid domain located immediately downstream from the secretion signal. TTCs are thought to stabilize effectors and have been postulated to maintain effectors in a partially unfolded state allowing them to be secretion competent. Chaperones are removed from their effectors before their translocation into host cells by the ATPase that is part of the Type III injectosome (2, 96). Not all effectors are known to utilize TTCs and it has been suggested that effectors that use TTCs are injected first into host cells. However, these research topics are controversial and little effort has been made to duplicate data obtained by one laboratory in another group. We still do not know how type III secretion signals or TTCs are recognized by the TTSS apparatus and whether there are distinct secretion and translocation signals (i.e., whether independent signals are needed for secretion out of the bacterial cell and injection into the eukaryotic cell). We would like to address the contribution of TTCs used by Pseudomonas syringae effectors and whether the TTSS apparatus recognizes TTCs to enable secretion of chaperoned effectors. This is critical for research on TTSSs because this is central to our understanding of how TTSSs deliver effector proteins into host cells. Our long-term goals are to understand how TTSSs inject effector proteins into host cells, to identify their eukaryotic targets, and to determine the effect these proteins have on host-microbe interactions. The objectives of this grant proposal are to seek to identify TTC binding sites on several P. syringae effectors and to determine how these effector regions contribute to the ability of effectors to be recognized, secreted, and translocated via the TTSS. The central hypothesis is that that TTCs help regulate type III secretion by stabilizing effectors and affecting regulation of the TTSS, and that specific TTSS apparatus proteins recognize the effectors instead of their TTCs. We formulated this hypothesis from closely following the literature on these topics, from our own published research, and recent preliminary data presented in this proposal, which support the central hypothesis in multiple ways. For example, we found that several effectors interact directly with a TTSS apparatus protein in yeast two hybrid experiments, while no TTCs were found to interact with any proteins of the TTSS apparatus suggesting that the apparatus recognizes effectors and not TTCs. Furthermore, we also found that one TTC interacts with several regulatory proteins of the TTSS, suggesting a role in the regulation of TTSSs. Once we understand how the TTSS apparatus recognizes protein substrates and how chaperones may aid in this secretion, the expectation is that this information will be exploited to circumvent the recognition process abrogating effector delivery or enhance recognition to favor the delivery of therapeutic proteins into plant or animal cells. We are prepared to undertake the proposed research because the PI has extensive experience in manipulating type III systems, and was one of the first to discover and work on P. syringae TTCs. She also has extensive experience using most of the proposed techniques. Collectively, this has positioned her extremely well to successfully carry-out the experiments described in this proposal in conjunction with her collaborators and the undergraduate students in her laboratory. The specific aims of this application are as follows: 1. Determine the contribution of TTC-binding domains to type III secretion and translocation of several effectors. We have carefully mapped the binding sites of several chaperones in their cognate effectors. Our working hypothesis is that deletion of the TTC binding sites will not disrupt secretion and translocation in the host of these effectors, suggesting that the secretion signal is contained within these effectors. 2. Identify in vitro and in vivo interactions between TTSS effectors, TTCs, and the TTSS apparatus. Our working hypothesis, based on our preliminary data, is that effectors and not their chaperones interact with TTSS gatekeeper(s). 3. Explore the TTSS regulation conferred by P. syringae TTCs. Our working hypothesis is that there is a hierarchy in the secretion of effectors whcih is affected by TTCs, and that a subset of P. syringae TTCs will be involved in the regulation of the TTSS. At the completion of these studies our expectation is that we will have a much better understanding of the contribution that TTCs make to the ability of P. syringae effectors to be secreted and translocated via the TTSS. This is important because it will shed much needed light on what makes a protein competent for traveling the TTSS pathway.